The role of duplication in the expression of a variable surface glycoprotein gene of Trypanosoma brucei

The variable surface glycoprotein (VSG) genes of Trypanosoma brucei have been classified into two groups depending upon whether or not duplication of the genes is observed when they are expressed. We report here the observation of duplication apparently linked to expression of the ILTaT 1.3 gene in the ETaR 1 trypanosome stock. In the ILTaR 1 stock, expression of the ILTaT 1.3 VSG did not involve a new duplication, but instead activation of a preexisting gene copy that had been apparently generated earlier by a duplication event analogous to that directly observed in the ETaR 1 trypanosomes. The results suggest that the well-characterised gene duplications found with other VSG genes are common to all VSG genes but are not directly responsible for controlling expression. All currently available data can be accommodated by a model that assumes that gene duplication and replacement occurs independently of antigenic switching.     

Distribution and multiplication of Ralstonia solanacearum strain race 1 biovar 4 in vegetable sweet potato cuttings

Ralstonia solanacearum (RS) race 1 biovar 4 (R1bv4), causal agent of bacterial wilt in vegetable sweet potato (VSP), is often latent in VSP vines and is important in introduction of the pathogen to newly planted fields. In this study, the effects of biological and environmental factors on the distribution and multiplication of R1bv4 in VSP tissues were examined. Based on stem-injection inoculation, the R1bv4strain of NC01 could cause 49.0% and 33.0% wilting on VSP cultivars TN71 and WS, respectively. The populations of NC01 in diseased TN71 and WS were 10⁸–10⁹ cfu/g tissue at 28th day after inoculation. On the other hand, the R1bv4 could not cause symptom in cultivars of TN57 and VSPSL-1 vine and the NC01 was confined to near the injection sites. Temperature tests indicated that NC01 could cause 28.0% and 14.0% wilting on cultivar TN71 at 28 and 20°C, respectively. Moreover, the populations of NC01in diseased plants were 1.6 × 10⁹ and 7.9 × 10⁸ cfu/g tissue at 28 and 20°C, respectively. The distribution of NC01 in VSP stem indicated that the isolation frequency of NC01 was lower than 31.0% in terminal shoots or erect stems and 45.0 to 100.0% in creeping stem after 8 wks planted in infested soil (10⁶ cfu/g soil). The results demonstrated that terminal shoots or erect stems were not common carrier for transmitting R1bv4. Furthermore, two R1bv4 strains, NC01 and HsinT01, were examined the ability for latent infection on cv. TN71. The results revealed that NC01 and HsinT01 showed different ability of latent infection on cultivar TN71. NC01 had lower percentage (46.8% and 45.1%) than HsinT01 (93.4% and 75.3%) at 20 and 28°C.     

Accumulation pattern of amino acid substitutions in protein evolution

Summary A simple method for the evolutionary analysis of amino acid sequence data is presented and used to examine whether the number of variable sites (NVS) of a protein is constant during its evolution. The NVSs for hemoglobin and for mitochondrial cytochrome c are each found to be almost constant, and the ratio between the NVSs is close to the ratio between the unit evolutionary periods. This indicates that the substitution rate per variable site is almost uniform for these proteins, as the neutral theory claims. An advantage of the present analysis is that it can be done without knowledge of paleontological divergence times and can be extended to bacterial proteins such as bacterial c-type cytochromes. It is suggested that the NVS of cytochrome c has been almost constant even over the long period (ca. 3.0 billion years) of bacterial evolution but that at least two different substitution rates are necessary to describe the accumulated changes in the sequence. This two clock interpretation is consistent with fossil evidence for the appearance times of photosynthetic bacteria and eukaryotes.     

Identification and partial characterization of a latent ATP,Mg-dependent protein phosphatase in rabbit skeletal muscle cytosol

Summary Fractionation of rabbit skeletal muscle cytosol on Aminohexyl-Sepharose has resulted in the identification of a latent ATP, Mg-dependent protein phosphatase whose catalytic subunit is in the active conformation, but is inhibited by the presence of more than one modulator unit. The partially purified enzyme is converted to an inactive, kinase F_A-dependent form upon incubation at 30°C unless modulator-specific polyclonal antibodies are added to the preparation. The immunoglobulins also relieve the inhibition which is responsible for the low basal phosphatase activity of the enzyme, and they counteract all of the heat-stable inhibitor activity present in the preparation. Addition of free catalytic subunit abolishes the inhibition of the latent enzyme in a dose-dependent way, but cannot prevent the inactivation process. The inactivated phosphatase and the original latent enzyme exhibit the same apparent M _r in sucrose density-gradient centrifugation (70 000) and in gel filtration (110 000).Abbreviations PMSF Phenylmethanesulphonyl Fluoride - TLCK L-l-chloro-3-(4-tosylamido)-7-amino2-heptanone-hydrochloride - TPCK L-l-chloro-3-(4-tosvlamido)-4-phenyl-2-butanone     

Characterization of chlorophyll fluorescence quenching in chloroplasts by fluorescence spectroscopy at 77 K II. ATP-dependent quenching

In intact, uncoupled type B chloroplasts from spinach, added ATP causes a slow light-induced decline ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 3}\text{min}$) of chlorophyll a fluorescence at room temperature. Fluorescence spectra were recorded after fast cooling to 77 K and normalized with fluorescein as an internal standard. Related to the fluorescence quenching at room temperature, an increase in Photosystem (PS) I fluorescence (F₇₃₅) and a decrease in PS II fluorescence (F₆₉₅) were observed in the low-temperature spectra. The change in the $\text{F}{}_{735}\text{F}{}_{695}$ ratio was abolished by the presence of methyl viologen. Fluorescence induction at 77 K of chloroplasts frozen in the quenched state showed lowered variable (F_v) and initial (F₀) fluorescence at 690 nm and an increase in F₀ at 735 nm. The results are interpreted as indicating an ATP-dependent change of the initial distribution of excitation energy in favor of PS I, which is controlled by the redox state of the electron-transport chain and, according to current theories, is caused by phosphorylation of the light-harvesting complex.     

Ammoniacal silver staining of proteins: mechanism of glutaraldehyde enhancement

When the conditions for detecting proteins by ammoniacal silver staining (B. R. Oakley, D. R. Kirsch, and N. R. Morris (1980) Anal. Biochem. 105, 361-363.) following gel electrophoresis were varied, it was noted that glutaraldehyde pretreatment was necessary for maximal staining, which could not be explained simply as the result of "fixation." Further studies indicated that glutaraldehyde enhancement of protein staining with this silver reagent was probably due to oxidation of the aldehyde groups by silver ions, resulting in metallic silver depositions within the gel which act as nucleation sites for additional metallic silver localization in the protein bands upon the addition of formaldehyde developer. This proposed mechanism is consistent with the Tollen's reaction, as well as some aspects of the photographic process. Consistent with this notion, silver-staining intensities are directly related to mole percentage lysine of various standard proteins.